To date, heterologous expression of herpesvirus thymidine kinase (HSV- TK) in combination with ganciclovir (GCV) has shown great clinical promise for a genetic therapy of different cancers. Initial clinical trial results have been overall quite promising, with a small percentage of patients having complete tumor repressions after HSV-TK/GCV treatments. However, the majority of patients have had only partial or minimal responses to the therapy. The goal of the work proposed herein is to increase the efficacy of this promising therapy by using a combination of rationally-designed mutations of HSV-TK to generate a GCV kinase (GCVK). Relative to currently used HSV-TKs, this GCVK will have improved phosphorylation activities for GCV and minimal thymidine kinase activities. Our hypothesis is that in anti-cancer gene therapies, these GCVKs will result in increased tumor cell killing, potential the in vivo immune response and potentially be safer due to lower required GCV doses. Towards this goal, several prototype GCVKs have already been characterized that possess either increased GCVK activity, reduced TK activity, or both, as evaluated by enzyme assays and cell culture expression. For this proposal, we will continue to design and characterize new and hybrid versions of these prototype GCVKs. The therapeutic efficacy of the most promising GCVKs with the desired enzymatic properties will be evaluated in colon tumor cell lines for their effect on GCV metabolism, cell killing and bystander effect. Further analysis of any increased therapeutic benefits of the new GCVKs will be evaluated in a multi-focal rat-tumor model of cancer. Successful demonstration of a therapeutic benefit in the proposed systems should provide the basis for clinical testing of these new GCVKs for treatment of metastatic liver disease and any genetic therapy currently employing the HSV-TK/GCV system.